U.S. patent number 5,238,947 [Application Number 07/705,015] was granted by the patent office on 1993-08-24 for synthetic piperidinediones with cytostatic activity.
This patent grant is currently assigned to Stereochemical Genetics, Inc., University of Georgia Research Foundation, Inc.. Invention is credited to Chung K. Chu, Lawrence B. Hendry, Virendra B. Mahesh.
United States Patent |
5,238,947 |
Hendry , et al. |
August 24, 1993 |
Synthetic piperidinediones with cytostatic activity
Abstract
The present invention are novel compounds of the formula:
##STR1## wherein R is OH, NH.sub.2, OW, or H; X is H, F, Cl, Br, I,
OH, OW, NO.sub.2 or NH.sub.2 ; Y is H, F, Cl, I, or Br; W is C(O)Z
or a C.sub.1 to C.sub.12 alkyl group; Z is an aliphatic or aromatic
group of from C.sub.1 to C.sub.12 ; X and Y can both vary within
the molecule; and if R is H, at least one of X or Y is not H. In a
preferred embodiment, R is OH or NH.sub.2. The most preferred
compound is (4-hydroxy-3-N-phenylacetylamino-2,6-piperidinedione),
in which R is OH, X is H and Y is H. These compounds have
cytostatic activity and insert stereochemically into DNA.
Inventors: |
Hendry; Lawrence B. (N.
Augusta, SC), Chu; Chung K. (Athens, GA), Mahesh;
Virendra B. (Augusta, GA) |
Assignee: |
University of Georgia Research
Foundation, Inc. (Athens, GA)
Stereochemical Genetics, Inc. (Augusta, GA)
|
Family
ID: |
27056321 |
Appl.
No.: |
07/705,015 |
Filed: |
May 21, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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508839 |
Apr 12, 1990 |
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Current U.S.
Class: |
514/328;
546/220 |
Current CPC
Class: |
C07D
211/88 (20130101) |
Current International
Class: |
C07D
211/00 (20060101); C07D 211/88 (20060101); A61K
031/00 (); C07D 211/40 () |
Field of
Search: |
;546/220 ;514/328 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Burzynski, et al., Chem. Abstract 105:164561w (1986). .
Burzynski, et al., Chem. Abstract 112:196233b (1990). .
Burzynski, et al., Drugs Exptl. Clin. Res. X(8-9), 611-619 (1984).
.
Burzynski, et al., Drugs Exptl. Clin. Res., Suppl. 1 XII, 11-16
(1986). .
Burzynski, Drugs Exptl. Clin. Res., Suppl. 1 XII, 17-24 (1986).
.
Burzynski, et al., Drugs Exptl. Clin. Res., Suppl. 1 XII, 47-55
(1986). .
Burzynski, Drugs of the Future 11(8), 679 (1986). .
Lee, et al., Chem. Abstract 107:17352s (1987). .
Lehner, et al., Drugs Exptl. Clin. Res., Suppl. 1 XII, 57-72
(1986). .
Xu, et al., Chem. Abstract 110:185528j (1989). .
Aguirre Ormaza, Vincente, Chem. Abstract 107:39628p (1987). .
Clissold, et al., Chem. Abstract 112:158062n (1990). .
De, et al., Chem. Abstract 89:179814f (1978). .
De, et al., Chem. Abstract 89:197241w (1978). .
Hibert, et al., Chem. Abstract 105:24193c (1986). .
Knabe, Drug Res. 39(II), 1379 (1989). .
Lee, et al., Chem. Abstract 102:105809y (1985). .
Otani, et al., Journal of Antibiotics XLII(5), 647 (1989). .
Shandala, et al., Chem. Abstract 110:75260h (1989). .
Sonoda, et al., Journal of Antibiotics XLL(11), 1607 (1989). .
Ashraf, et al., Drugs Exptl. Clin. Res., Suppl 1 XII, 37-45 (1986).
.
Burzynski, Chem. Abstract 90:37144j (1979). .
Burzynski, et al., Chem. Abstract 101:204012u (1984). .
Wintrobe, et al.; (Harrison's) Principles of Internal Medicine 7th
Ed pp. 577-587, 1974..
|
Primary Examiner: Cintins; Marianne M.
Assistant Examiner: Scalzo; Catherine
Attorney, Agent or Firm: Kilpatrick & Cody
Parent Case Text
This application is a continuation of application Ser. No.
07/508,839 filed on apr. 12, 1990, now abandoned.
Claims
We claim.
1. A compound of the formula: ##STR4## or its pharmaceutically
acceptable salt.
2. A pharmaceutical composition comprising an effective cytostatic
amount for humans of 3-2,6-dione in an pharmaceutically acceptable
carrier.
3. The pharmaceutical composition of claim 2 wherein the
pharmaceutical carrier is for topical administration.
4. The pharmaceutical composition of claim 2 where the
pharmaceutical carrier is for systemic administration.
5. A method of inhibiting cell growth in a patient in need thereof
comprising providing an effective amount of a compound of the
formula ##STR5## or its pharmaceutically acceptable salt.
Description
BACKGROUND OF THE INVENTION
This invention is in the area of organic chemistry, and
specifically relates to new piperidinedione derivatives with
cytostatic properties.
A tumor is an unregulated, disorganized proliferation of cell
growth. A tumor is malignant, or cancerous, if it has the
properties of invasiveness and metastasis. Invasiveness refers to
the tendency of a tumor to enter surrounding tissue, breaking
through the basal laminas that define the boundaries of the
tissues, thereby often entering the body's circulatory system.
Metastasis refers to the tendency of a tumor to migrate to other
areas of the body and establish areas of proliferation away from
the site of initial appearance.
Cancer is now the second leading cause of death in the United
States, Europe, and Japan, resulting in approximately 1,000,000
deaths annually in these countries. In the United States alone,
each year over one million people are diagnosed with cancer, and
over 500,000 people die from the disease. The number of newly
diagnosed cancerous growths in patients in the United States is
growing at a rate of 3% a year.
Cancer is not fully understood on the molecular level. It is known
that exposure of a cell to a carcinogen such as certain viruses,
certain chemicals, or radiation, leads to DNA alteration that
activates han "oncogene." Oncogenes are initially normal genes
(called prooncogenes) that by mutation or altered context of
expression become transforming genes. The products of transforming
genes cause inappropriate cell growth. More than twenty different
normal cellular genes can become oncogenes by genetic alteration.
Transformed cells differ from normal cells in many ways, including
cell morphology, cell-to-cell interactions, membrane content,
cytoskeletal structure, protein secretion, gene expression and
mortality (transformed cells can grow indefinitely).
All of the various cell types of the body can be transformed into
malignant cells. The most frequent tumor site is lung, followed by
colorectal, breast, prostate, bladder, pancreas, and then
ovary.
Cancer is now treated with one or a combination of three types of
therapies: surgery, radiation, and chemotherapy Surgery involves
the bulk removal of diseased tissue. While surgery is sometimes
effective in removing tumors located at certain sites, for example,
in the breast, colon, and skin, it cannot be used in the treatment
of tumors located in other areas, such as the backbone, nor in the
treatment of disseminated neoplastic conditions such as
leukemia.
Chemotherapy now represents less than 4% of the total expenditures
on the treatment of cancer. Chemotherapy involves the disruption of
cell replication or cell metabolism. It is used most often in the
treatment of leukemia and breast, lung, and testicular cancer.
There are four major classes of chemotherapeutic agents currently
in use for the treatment of cancer; anthracyclines, alkylating
agents, antiproliferatives, and hormonal agents. A variety of
methods exist to attempt to identify new antineoplastic
chemotherapeutic agents, including random screening of compounds,
preparation of analogs of active compounds, computer or physical
modeling, and combinations of these techniques. None of these
methods, however, have yet identified the optimal chemotherapeutic
agent for neoplastic diseases. U.S. Pat. No. 4,461,619 to Hendry et
al., discloses a method to determine the relationship of chemical
structure to biological activity based on the topology and
physicochemical properties of "cavities" or "artificial constructs"
in double stranded DNA, double stranded RNA, or double stranded
DNA-RNA. While modeling can be very helpful in chemotherapy
research, it cannot predict whether a target compound will pass
through the cell wall, whether it is stable in vivo generally or in
the cytoplasm specifically, or whether the therapeutic index is
appropriate for clinical use of the drug.
Burzynski has proposed that the human organism is equipped with a
corrective system that can reprogram the growth of newly developed
neoplastic cells to transform them back into normal cells. He has
isolated a number of medium sized peptides, referred to as
antineoplastons, that are produced by the body to protect it
against the development of neoplastic growth by a nonimmunological
process that does not significantly inhibit the growth of normal
tissue. The most potent antineoplaston isolated by Burzynski is
3-[N-phenylacetylaminopiperidine]-2,6-dione (referred to below as
A10). Antineoplastons are described in U.S. Pat. No. 4,444,890,
entitled "Testing Procedure to Aid Diagnosis of Cancer and Evaluate
the Progress of Cancer Therapy"; U.S. Pat. No. 4,593,038, entitled
"Topical Use of 3-Phenylacetylamino-2,6-Piperidinedione for
Treatment of Skin Wrinkles and Hyperpigmentation"; and U.S. Pat.
Nos. 4,558,057, 4,559,325 and 4,470,970, entitled "Purified
Antineoplaston Fractions and Methods of Treating Neoplastic
Disease." According to these patents, administration of
antineoplastons to cancer patients has resulted in symptomatic
improvement in 93% of the patients treated. A remission of the
tumor was noted in about 45% of the patients
The initial hydrolysis product and biological degradation product
of A10 is phenylacetylglutamine, which is produced in vivo from
phenylacetic acid and glutamine. In fact, A10 may be cyclized from
phenylacetylglutamine in vivo. Markaverich, et al., report that a
compound structurally related to phenylacetic acid, methyl
p-hydroxyphenylacetate, inhibits MCF-7 human breast cancer cells in
vitro. Markaverich, et al., J. of Biol. Chem. 263(15), 7203
(1988).
Hendry has shown that A10 fits in a stereochemical manner between
base pairs of double stranded DNA. Hendry, L.B., et al., "Modeling
Studies Suggest the Modified Dipeptide Analog
Phenylacetylamino-2,6-piperidinedione may interact with DNA,"
Advances in Experimental and Clinical Chemotheraoy. 15th
International Congress of Chemotherapy, Istanbul, Turkey, 1987.
Specifically, A10 is capable of forming a stereospecific hydrogen
bond between the imino proton of the piperidinedione ring and the
phosphate oxygen on the DNA backbone. A10 does not bind covalently
to DNA, which may explain why the compound is cytostatic and not
cytotoxic. The acute toxicity of A10 in mice is between 1.35 and
10.33 g/kg. AIO has been administered without serious side effects
at a dosage of up to 10 grams per day to humans suffering from
cancer.
While A10 is a useful drug in the treatment o neoplastic diseases,
there is a need for new cytostatic agents that may be more
effective in stimulating tumor remission, and that may be effective
when administered in lower dosages. The tragic number of deaths
that occur each year from this disease accentuates the urgency of
this need.
In light of the above, it is clear that there is a strong need for
new cytostatic agents that can effectively insert stereochemically
into DNA.
Therefore, it is an object of the present invention to provide a
compound that has a cytostatic effect on cancer cells.
It is a further object of the present invention to provide a
compound that can insert stereochemically into DNA.
SUMMARY OF THE INVENTION
The present invention is a compound with cytostatic activity of the
formula: ##STR2## wherein R is OH, NH.sub.2, OW, or H; X is H, F,
Cl, Br, I, OH, OW, NO.sub.2 or NH.sub.2 ; Y is H, F, Cl, I, or Br;
W is C(O)Z or a C.sub.1 to C.sub.12 alkyl group; Z is an aliphatic
or aromatic group of from C.sub.1 to C.sub.12 ; X and Y can both
vary within the molecule; and if R is H, at least one of X or Y is
not H.
In a preferred embodiment, R is OH or NH.sub.2. The most preferred
compound is (4-hydroxy-3-N-phenylacetylamino-2,6-piperidinedione),
in which R is OH, X is H and Y is H.
These compounds exhibit cytostatic activity and inhibit the uptake
of radioactive thymidine in a variety of neoplastic cell lines. The
preferred compound,
4-hydroxy-3-N-phenylacetylamino-2,6-piperidinedione, has
significant cytostatic activity against prolactin stimulated Nb2
lymphoma cells (a T cell derived lymphoma), MCF-7 cells (estrogen
sensitive cells), and mouse lymphoma (YAK) cells. "Cytostatic
activity" as used herein refers to the ability of a compound to
inhibit cell growth or replication. In contrast, "cytotoxic
activity" refers to the ability of a compound to kill cells.
Cytostatic agents typically cause reversible chemical changes in
cells. An example of a reversible change is the formation of
anionic bond through ionic bonds. Cytotoxic compounds typically
cause irreversible changes in cells. An example of an irreversible
change is the formation of a covalent bond.
Modeling studies suggest that
4-hydroxy-3-N-phenylacetylamino-2,6-piperidinedione (referred to
below as p-OH-A10) fits easily between base pairs of DNA to form a
complex with significantly lower energy than either structure
alone. It has now been discovered that the compounds described
herein in which R is a proton donating group such as hydroxyl or
amino have significantly greater cytostatic effect than A10.
Further, it has been discovered that the preferred compounds of
this invention are capable of binding to both strands of the DNA,
whereas A10 is only capable of binding to one of the strands of
DNA. Thymidine uptake studies indicate that the compounds inhibit
DNA synthesis.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic illustrating a method of preparation of the
cytostatic piperidinediones of the present invention. The aryl
groups in the piperidinedione compounds are abbreviated as follows:
a, 4-HO-C.sub.6 H.sub.4 ; b, 2-Cl,6-F-C.sub.6 H.sub.3 ; c,
2-F-C.sub.6 H.sub.4 ; d, 3-F-C.sub.6 H.sub.4 ; e, 4-F-C.sub.6
H.sub.4 ; f, 2,6-di-F-C.sub.6 H.sub.3 ; g, 4-CH.sub.3 O-C.sub.6
H.sub.4 ; h, 4-Cl-C.sub.6 H.sub.4 ; i, 4-Br-C.sub.6 H.sub.4 ; j,
4-CF.sub.3 -C.sub.6 H.sub.4. The following reagents were used: step
(i), N,N-dicyclohexylcarbodiimide (DCC) in acetonitrile; step (ii),
NaHCO.sub.3 in acetonitrile/H.sub.2 O (2/1 ratio); step (iii), DCC
in dimethylformamide (DMF); and step (iv), DMF.
FIG. 2 is a bar chart graph indicating the inhibition of prolactin
stimulated Nb2 lymphoma cell growth by cytostatic piperidinediones.
Equivalent amounts (1 mg/ml) of A10 or A10 analogs were introduced
simultaneously with prolactin (PRL) (0.4 ng/ml) to quiescent Nb2
lymphoma cells. Cell counts were made 48 hours later. The following
abbreviations were used for the compounds tested: HQH-1-45-28,
3-[N-2-Chloro,6-fluorophenylacetylaminopiperidine]-2,6-dione;
HQH-1-44-11, 3-[N-2-fluorophenylacetylaminopiperidine]-2,6-dione;
HQH-1-41-27,
3-[N-2,6-difluorophenylacetylaminopiperidine]-2,6-dione;
HQH-1-36-36, 3-[N=4-hydroxyphenylacetylaminopiperidine]-2,6-dione;
HQH-1-51-29, 3-[N-4-fluorophenylacetylaminopiperidine]-2,6-dione;
and HQH-2-48-30,
3-[N-3-fluorophenylacetylaminopiperidine]-2,6-dione.
FIG. 3 is a graph indicating the inhibitory effect of
3-[N-4-hydroxyphenylacetylaminopiperidine]-2,6-dione (A10) and
3-[N-phenylacetylaminopiperidine]-2,6-dione (p-OH-A10) on MCF-7
cells growing in log phase.
FIG. 4 is a bar chart graph indicating the inhibition of mouse
lymphoma (YAK) cell proliferation by the cytostatic
piperidinediones of the present invention
(PAG=phenylacetylglutamine; PA=phenylacetic acid). The compound
numbers are as indicated in FIG. 2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a compound with cytostatic activity of the
formula: ##STR3## wherein R is OH, NH.sub.2, OW, or H; X is H, F,
Cl, Br, I, OH, OW, NO.sub.2 or NH.sub.2 ; Y is H, F, Cl, I, or Br;
W is C(O)Z or a C.sub.1 to C.sub.12 alkyl group; Z is an aliphatic
or aromatic group of from C.sub.1 to C.sub.12 ; X and Y can both
vary within the molecule; and if R is H, at least one of X or Y is
not H.
Active compounds include
3-[N-4-hydroxyphenylacetylaminopiperidine]-2,6-dione,
3-[N-4-hydroxy-3-fluorophenylacetylaminopiperidine]-2,6-dione,
3-[N-4-hydroxy-3-chlorophenylacetylaminopiperidine]-2,6-dione,
3-[N-3,4-dihydroxyphenylacetylaminopiperidine]-2,6-dione,
3-[N-3-amino-4-hydroxyphenylacetylaminopiperidine]-2,6-dione,
3-[N-4-aminophenylacetylaminopiperidine]-2,6-dione,
3-[N-4-methoxy-3-hydroxyphenylacetylaminopiperidine]-2,6-dione,
3-[N-4-amino-3-fluorophenylacetylaminopiperidine]-2,6-dione,
3-[N-2-fluoro-4-hydroxyphenylacetylaminopiperidine]-2,6-dione,
3-[N-2-fluorophenylacetylaminopiperidine]-2,6-dione,
3-[N-4-acyloxyphenylacetylaminopiperidine]-2,6-dione, and
3-[N-4-alkoxyphenylacetylaminopiperidine]-2,6-dione.
In a preferred embodiment, the active compound has a proton
donating group (such as hydroxyl or amino) in the 4-position of the
aromatic ring and an electron withdrawing or proton donating group
in the 3-position of the aromatic ring. The 2-position of the
aromatic ring is preferably unsubstituted (Y=H) or substituted with
a small group that does not hinder free rotation of the aromatic
ring.
These compounds are synthetic derivatives of the naturally
occurring antineoplaston, A10. It has been demonstrated through
thymidine incorporation studies that A10 significantly inhibits DNA
synthesis (16% at 20 hours; 30% at 40 hours, p<0.01). Modeling
has indicated that A10 can insert into DNA and form a stabilizing
hydrogen bond through its imide group to a phosphate oxygen on one
strand of the double stranded DNA.
It has been now been established through thymidine studies that the
compounds described herein also significantly inhibit DNA
synthesis. Further, it has been discovered that the novel compounds
described herein that have a proton donating group, such as
hydroxyl or amino, in the 4-position of the aromatic ring have
significantly more cytostatic activity than those piperidinedione
compounds without proton donating groups in these positions. It is
postulated that the increased activity is due to the ability of the
proton donating group at the 4-position of the molecule to form a
stabilizing stereospecific hydrogen bond with a phosphate oxygen on
the nucleic acid strand opposite to that hydrogen bonded to the
imide group of the synthetic piperidinedione. These active
compounds thus appear to be capable of securing themselves to both
strands of DNA in the double helix, resulting in a complex of
significantly lower energy than a complex of double stranded DNA
with an piperidinedione that is capable of hydrogen bonding to only
one strand of the helix, such as A10. As a result, these compounds
are expected to have greater therapeutic efficacy and lower
toxicity than A10.
Cytostatic agents are useful to moderate the growth of
proliferative cells such as neoplastic cells in cell culture or
animal models for purposes of research in the area of proliferative
diseases. Cytostatic agents are also useful in the study of the
bonding patterns and consequent activity of ribonucleic acids.
Cytostatic agents may also have a pharmaceutical use as
antineoplastic agents in vivo.
The present invention will be further understood with reference to
the following non-limiting examples describing the synthesis,
activity, and preparation of pharmaceutical compositions of these
compounds.
I. Method of Preparation of Cytostatic Piperidinediones
The cytostatic piperidinedione derivatives can be prepared by
condensation of the appropriate phenylacetic acid derivative with
L-glutamine to produce the corresponding phenylacetylglutamine
derivative, that is then intramolecularly cyclized to the desired
3-(N-phenylacetylamino)-2,6-piperidinedione. The condensation
reaction is facilitated by prior activation of the phenylacetic
acid derivative with a reagent such as N-hydroxysuccinimide in the
presence of DCC (N,N-dicyclohexylcarbodiimide),
2-mercaptothiazoline in the presence of DCC, or DCC alone. The
phenylacetylglutamine derivative is also preferably activated
before cyclization by reaction with N-hydroxysuccinimide in the
presence of DCC or by reaction with 1,1'-carbonyldiimidazole. These
reactions are described in more detail in Burzynski, Drugs of the
Future, 10(2), 1003 (1985).
Desired derivatives of phenylacetic acid can be purchased
commercially or prepared synthetically by methods known to those
skilled in the art according to well established rules of
electrophilic and nucleophilic aromatic substitution. For example,
4-hydroxyphenylacetic acid, which is commercially available from
Aldrich Chemical Company, Inc., can be nitrated with dilute
HNO.sub.3 to produce 4-hydroxy-3-nitrophenylacetic acid, that is
used as is in the next step of reaction. Alternatively, the nitro
group in 4-hydroxy-3-nitrophenylacetic acid can be reduced to the
corresponding amine and then reacted with sodium nitrite in acid to
form the diazonium salt, that can be converted into a wide range of
functional groups, including chloro (CuCl), fluoro (HBF.sub.4),
bromo (CuBr) and hydroxyl (H.sub.2 SO.sub.4). Phenylacetic acid can
alternatively be nitrated in the 4-position to produce
4-nitrophenylacetic acid, that is used as is in the reaction or
converted to the diazonium salt and derivatized. The nitro group
can be reduced to the corresponding amino group as a final step of
reaction by methods known to those skilled in the art, including
catalytic hydrogenation with palladium on carbon.
Prodrugs of the hydroxyl or amino derivatives of
3-N-phenylacetylamino-2,6-piperidinedione can be prepared by
alkylation or acylation of the hydroxyl or amino moieties according
to established methods. These protecting groups can be cleaved in
vivo or in vitro by the appropriate enzyme, generating the active
compound.
In FIG. 1, a general reaction scheme is illustrated for the
preparation of the cytostatic piperidinediones. As shown, the
substituted phenylacetic acids 1 were reacted with
N-hydroxysuccinimide to produce the corresponding
N-hydroxysuccinimide esters 2, that were reacted with L-glutamine
to form the phenylacetylglutamine derivatives 3. Compound 3 was
then reacted with N-hydroxysuccinimide and cyclized to form the
substituted 3-N-phenylacetylamino-2,6-piperidinedione derivatives
5. Detailed experimental procedures are provided in Examples 1-4.
(The numbering scheme for the compounds described in these Examples
is specified in FIG. 1.) Physical and NMR data for the
N-hydroxysuccinimide esters of substituted phenylacetic acids are
provided in Tables 1 and 2, respectively. NMR data for the
substituted phenylacetyl-L-glutamines and
substituted-3-N-phenylacetylamino-2,6-piperidinediones are provided
in Tables 3 and 4, respectively.
In the following working examples, melting points were determined
on a Thomas Hoover capillary apparatus and are uncorrected. .sup.1
N NMR spectra were recorded on a JEOL FX 90Q fourier transform
spectrometer for the 90-MHz .sup.1 H NMR spectra, using Me.sub.4 Si
as internal standard: chemical shifts are reported in parts per
million (.delta.) and signals are quoted as s (singlet), d
(doublet), t (triplet), q (quartet), or m (multiplet). UV spectra
were obtained on a Beckman DU-7 spectrophotometer. Thin layer
chromatography was performed on Uniplates (silica gel) purchased
from Analtech Co. Elemental analyses were performed by Atlantic
Microlab Inc., Norcross, GA or Galbrait Laboratories, Inc.,
Knoxville, Tenn.
TABLE 1
__________________________________________________________________________
Physical Constants and Microanalysts of Compounds Compd. % C % H %
N % F % Cl Br % (Overall .lambda..sub.max UV(nm) Calcd./ Calcd./
Calcd./ Calcd./ Calcd./ Calcd./ yield) mp .degree.C. (CH.sub.3 OH)
Formula Anal. Found Found Found Found Found Found
__________________________________________________________________________
2a 138-140 C.sub.12 H.sub.11 NO.sub.5 C, H, N 57.83 4.45 5.62 57.90
4.46 5.58 2b 109.5-111 C.sub.12 H.sub.9 FClNO.sub.4 C, H, N 50.45
3.18 4.90 50.55 3.23 4.83 2c 95-96 C.sub.12 H.sub.10 FNO.sub.4 C,
H, N 57.37 4.01 5.58 57.22 3.99 5.53 2d 90-91.5 C.sub.12 H.sub.10
FNO.sub.4 C, H, N 57.37 4.01 5.58 57.45 4.00 5.58 2e 100.5-101.5
C.sub.12 H.sub.10 FNO.sub.4 C, H, N 57.37 4.01 5.58 57.26 4.03 5.59
2f 110-111 C.sub.12 H.sub.9 F.sub.2 NO.sub.4 C, H, N 53.54 3.37
5.20 53.56 3.41 6.19 2g 108.5-109.5 C.sub.13 H.sub.13 NO.sub.5 C,
H, N 59.31 4.98 5.32 59.22 4.99 5.30 2h 135-136 C.sub.12 H.sub.10
ClNO.sub.4 C, H, N, Cl 53.84 3.77 5.33 13.24 53.94 3.76 5.19 13.18
2i 139-141 C.sub.12 H.sub.10 BrNO.sub.4 C, H, N, Br 46.17 3.23 4.49
25.60 46.22 3.24 4.42 25.66 2j 120-121 C.sub.13 H.sub.10 F.sub.3
NO.sub.4 C, H, N, F 51.84 3.35 4.65 18.92 52.16 3.33 4.49 18.72 5a
(17.1%) 172-174 229 C.sub.13 H.sub.14 N.sub.2 O.sub.4 C, H, N 59.53
5.38 10.68 59.45 5.43 10.67 5b (42.5) 203-204.5 216 C.sub.13
H.sub.12 FClN.sub.2 O.sub.3 C, H, N 52.27 4.05 9.38 52.37 4.06 9.29
5c (26.5) 185-188.5 212.5 C.sub.13 H.sub.13 FN.sub.2 O.sub.3 C, H,
N 59.08 4.97 10.60 59.19 4.95 10.51 5d (20.3) 180-181 209.7
C.sub.13 H.sub.13 FN.sub.2 O.sub.3 C, H, N 59.08 4.97 10.60 59.16
4.99 10.55 5e (21.5) 190-191 210 C.sub.19 H.sub.13 FN.sub.2 O.sub.3
C, H, N 59.08 4.96 10.60 59.03 5.01 10.62 5f (38.5) 188-189 212.5
C.sub.13 H.sub.12 F.sub.2 N.sub.2 O.sub.3 C, H, N 55.32 4.29 9.93
55.24 4.30 9.90 5g (32.9) 193-194 227.9 C.sub.14 H.sub.16 N.sub.2
O.sub.4 C, H, N 60.85 5.85 10.14 60.86 5.87 10.20 5h (40.3)
207.5-208.5 225 C.sub.13 H.sub.13 ClN.sub.2 O.sub.3 C, H, N, Cl
55.62 4.68 9.98 12.63 55.66 4.66 9.93 12.69 5i (40.7) 213-214 228.9
C.sub.13 H.sub.13 BrN.sub.2 O.sub.3 C, H, N, Br 48.02 4.04 8.62
24.57 47.92 4.07 8.57 24.67 5j (41.8) 177-178 227 C.sub.14 H.sub.13
F.sub.3 N.sub.2 O.sub.3 C, H, N, F 53.60 4.18 8.92 18.14 53.50 4.07
8.84 18.69
__________________________________________________________________________
TABLE 2
__________________________________________________________________________
.sup.1 H-NMR Signals Observed for N-Hydroxysuccinimide Esters of
Substituted Phenylacetic Acids (2a-2j) (.delta. ppm downfield from
TMS, in DMSO-d.sub.6) Compd. Aromatic H Other Signals
__________________________________________________________________________
2a 6.95(4H, d-d, J=8.5, 35.7Hz) 2.79(4H, s, COCH.sub.2 CH.sub.2
CO), 3.92(2H, s, CH.sub.2 COO), 9.38(1H, s, OH) 2b 7.40(3H, m)
2.80(4H, s, COCH.sub.2 CH.sub.2 CO), 4.22(2H, d, J=1.47Hz, CH.sub.2
COO) 2c 7.13-7.55(4H, m) 2.80(4H, s, COCH.sub.2 CH.sub.2 CO),
4.15(2H, s, CH.sub.2 COO) 2d 7.05-7.55(4H, m) 2.81(4H, s,
COCH.sub.2 CH.sub.2 CO), 4.17(2H, s, CH.sub.2 COO) 2e 7.08-7.48(4H,
m) 2.81(4H, s, COCH.sub.2 CH.sub.2 CO), 4.12(2H, s, CH.sub.2 COO)
2f 7.08-7.56(3H, m) 2.81(4H, s, COCH.sub.2 CH.sub.2 CO), 4.16(2H,
s, CH.sub.2 COO) 2g 7.09(4H, d-d, J=7.03, 22.27Hz) 2.80(4H, s,
COCH.sub.2 CH.sub.2 CO), 3.74(3H, s, OCH.sub.3), 4.00(2H, s,
CH.sub.2 COO) 2h 7.41(4H, m) 2.81(4H, s, COCH.sub.2 CH.sub.2 CO),
4.14(2H, s, CH.sub.2 COO) 2i 7.44(4H, d-d, J=8.21, 24.31Hz)
2.28(4H, s, COCH.sub.2 CH.sub.2 CO), 4.12(2H, s, CH.sub.2 COO) 2j
7.67(4H, d-d, J=8.49, 15.82Hz) 2.81(4H, s, COCH.sub.2 CH.sub.2 CO),
4.23(2H, s, CH.sub.2 COO)
__________________________________________________________________________
TABLE 3
__________________________________________________________________________
.sup.1 H-NMR Signals Observed for (Substituted
Phenylacetyl)-L-Glutamine (.delta. ppm downfield from TMS, in
DMSO-d.sub.6) Compd. Aromatic H ArCH.sub.2 CON Other Signals
__________________________________________________________________________
3a 6.85(4H, d-d, J=8.8, 32.2Hz) 3.31(2H, s) 2.41(4H, s, CH.sub.2
CH.sub.2), 3.46(1H, b, NC .sub.-- H(CH.sub.2 CH.sub.2)COOH),
3.80(1H, b, CONH), 7.20(1H, s, COOH), 7.28(1H, s, OH), 7.40(2H, s,
CONH.sub.2) 3b 7.12-7.58(3H, m) 3.71(2H, s) 2.50(4H, s, CH.sub.2
CH.sub.2), 3.45(1H, b, NC .sub.-- H(CH.sub.2 CH.sub.2)COOH),
3.85(1H, b, CONH), 6.59(2H, b, CONH.sub.2), 12.27(1H, b, COOH) 3d
7.03-7.55(4H, m) 3.52(2H, s) 1.95(2H, s, C .sub.-- H.sub.2
CH.sub.2), 2.41(2H, s, CH.sub.2 C .sub.-- H.sub.2), 3.24(1H, b, NC
.sub.-- H(CH.sub.2 CH.sub.2)COOH), 3.80(1H, b, CON 6.60(2H, b,
CONH.sub.2), 7.65(1H, b, COOH) 3g 7.00(4H, d-d, J=7.62, 30.18Hz)
3.38(2H, s) 1.92(2H, m, C .sub.-- H.sub.2 CH.sub.2), 2.49(2H, m,
CH.sub.2 C .sub.-- H.sub.2), 3.74(3H, s, OCH.sub.3), 3.77 (1H, m,
NC .sub.-- H(CH.sub.2 CH.sub.2)COOH), 3 6.55(2H, m, CONH2),
7.33(1H, d, J=7.03, CONH), 7.37(1H, m, COOH) 3h 7.31(4H, m)
3.47(2H, s) 2.01(4H, b, CH.sub.2 CH.sub.2), 4.10(1H, m, NC .sub.--
H(CH.sub.2 CH.sub.2)COOH), 6.74(2H, m, CONH.sub.2), 7.30(1H, m,
COOH), 8.35(1H, m, CONH) 3i 7.34(4H, d-d, J=8.21, 23.73Hz) 3.45(2H,
s) 1.91(4H, b, CH2CH.sub.2), 4.13(1H, m, NC .sub.-- H(CH.sub.2
CH.sub.2)COOH), 6.74(2H, m, CONH.sub.2), 7.43(1H, m, COOH),
8.42(1H, m, CONH) 3j 7.57(4H, d-d, J=8.20, 16.7Hz) 3.59(2H, s)
1.97(4H, b, CH.sub.2 CH2), 4.15(1H, m, NC .sub.-- H(CH.sub.2
CH.sub.2)COOH), 6.75(2H, s, CONH.sub.2), 7.26(1H, m, COOH),
8.44(1H, m, CONH)
__________________________________________________________________________
TABLE 4
__________________________________________________________________________
1H-NMR Signals Observed for
Substituted-3-N-Phenylacetyl-Amino-2,6-Piperid inedione (5a-5j)
(.delta. ppm downfield from TMS, in DMSO-d.sub.6) Compd. Aromatic H
ArCH.sub.2 CON CONH-ring (CH.sub.2 CH.sub.2)CH*CO CH.sub.2 CH.sub.2
ring-NH Other
__________________________________________________________________________
Signal 5a 6.88(4H, d-d, 3.37(2H, s) 8.28(1H, d, J=9Hz) 4.36(1H, q,
J=9Hz) 1.85(2H, m) 9.20(1H, 10.75(1H, s, J=8.9, 32.2Hz) **4.49(1H,
t, J=9Hz) 2.50(2H, m) OH) 5b 7.30(3H, m) 3.20(2H, s) 8.46(1H, d,
J=8.2Hz) 4.50(1H, q, J=8.2Hz) 1.95(2H, m) 10.80(1H, s) **4.53(1H,
t, J=8.2Hz) 2.55(2H, m) 5c 7.22(4H, m) 3.56(2H, s) 8.43(1H, d,
J=9.3Hz) 4.54(1H, q, J=9.3Hz) 1.93(2H, m) 10.79(1H, s) **4.52(1H,
t, J=9.3Hz) 2.50(2H, m) 5d 7.20(4H, m) 3.52(2H, s) 8.46(1H, d,
J=8.2Hz) 4.56(1H, q, J=8.2Hz) 1.94(2H, m) 10.80(1H, s) **4.52(1H,
t, J=8.2Hz) 2.57(2H, m) 5e 7.21(4H, m) 3.45(2H, s) 8.41(1H, d,
J=7.9Hz) 4.54(1H, q, J=7.9Hz) 1.92(2H, m) 10.79(1H, s) **4.53(1H,
t, J=7.9Hz) 2.70(2H, m) 5f 7.18(3H, m) 3.58(2H, s) 8.47(1H, d,
J=8.0Hz) 4.53(1H, q, J=8.0Hz) 1.93(2H, m) 10.80(1H, s) **4.50(1H,
t, J=8.0Hz) 2.45(2H, m) 5g 7.02(4H, d-d, 3.40(2H, s) 8.32(1H, d,
J=7.91Hz) 4.53(1H, q, J=7.91Hz) 1.91(2H, m) 10.77(1H, 3.72(3H, s,
J=8.79, 31.34Hz) **4.52(1H, t, J= 2.54(2H, m) OCH.sub.3 7.91Hz) 5h
7.33(4H, s) 3.49(2H, s) 8.42(1H, d, J=7.91Hz) 4.55(1H, q, J=8.01Hz)
1.90(2H, m) 10.78(1H, s) **4.52(1H, t, J= 2.54(2H, m) 7.95Hz) 5i
7.32(4H, d-d, 3.47(2H, s) 8.43(1H, d, J=8.21Hz) 4.53(1H, q,
J=8.6Hz) 1.91(2H, m) 10.79(1H, s) J=8.50, 23.43Hz) **4.51(1H, t,
J=8.6Hz) 2.54(2H, m) 5j 7.58(4H, d-d, 3.62(2H, s) 8.50(1H, d,
J=7.91Hz) 4.54(1H, q, J=7.95Hz) 1.92(2H, m) 10.79(1H, s) J=8.49,
15.86Hz) **4.55(1H, t, J=7.95Hz) 2.54(2H, m)
__________________________________________________________________________
**after exchanged with D.sub.2 O
Example 1:
General Procedure for the Preparation of N-Hydroxysuccimide Esters
of substituted Phenylacetic Acids.
The following is a detailed description for the preparation of the
N-hydroxysuccinimide ester of 2-chloro-6-fluorophenylacetic acid
2b. The compounds 2a, 2c-2j were prepared as described below from
the appropriate substituted phenylacetic acids (1a, 1c-1j),
respectively.
A solution of N-hydroxysuccinimide (5 g, 42 mmol) in 30 mL of
anhydrous acetonitrile (dried over 4 .ANG. molecular sieve) was
mixed with a solution of 2-chloro-6-fluoro-phenylacetiC acid (1b,
7.92 g, 42 mmol, purchased from Aldrich Chemical Company, Inc., in
50 mL of anhydrous acetonitrile. The clear solution was cooled in
an ice bath and N,N-dicyclohexylcarbodiimide (8.66 g, 42 mmol) in
50 mL of anhydrous acetonitrile was added to the mixture with
stirring. The reaction mixture was stirred at ambient temperature
for 23 hours, until thin layer chromatography indicated the
disappearance of the starting material (ethyl acetate/hexanes,
1/1). The colorless precipitate obtained (dicyclohexylurea) was
filtered off. The filter cake was washed with acetonitrile and the
combined filtrate was then evaporated under vacuum to give a
colorless solid (2b, 12.57 g) that was used for next reaction
without further purification. Analytical samples were prepared by
preparative thin layer chromatography using ethyl acetate/hexanes
(1/1).
Example 2:
General Procedure for the Preparation of Substituted
Phenylacetyl-L-Glutamines.
To a solution of the N-hydroxysuccimide ester of
2-chloro-6-fluoro-phenyl acetic acid (2b, 12.5 g, 42 mmol) in 120
mL of acetonitrile was added a solution of L-glutamine (6.14 g, 42
mmol) in a mixture of 170 mL of water and 340 mL o acetonitrile
containing sodium bicarbonate (7 g, 84 mmol). The mixture was
stirred at ambient temperature for 24 hours. The organic layer was
separated, and the aqueous layer then extracted with 100 mL
acetonitrile. The combined organic layer was evaporated under
vacuum to give 3b (15.3 g) as colorless solid. A small amount of
the sample was purified by thin layer chromatography
(chloroform/methanol, 10.1) for spectroscopic characterization.
The compounds of 3a, 3c-3j were prepared as described above from
2a, 2c-2j, respectively.
Example 3:
General Procedure for the Preparation of N-Hydroxysuccinimide
Esters of (Substituted Phenylacetyl)-L-Glutamine.
A solution of N-hydroxysuccinimide (5 g, 42 mmol) in 50 mL of
anhydrous dimethylformamide (dried over 4.ANG. molecular sieve) was
added to the solution of 2-chloro-6-fluoro-phenylacetyl-L-glutamine
(3b, 15.3 g, 42 mmol) in 300 mL of anhydrous DMF.
N,N-Dicyclohexylcarbodiimide (8.66 g, 42 mmol) was added to the
mixture with stirring. The mixture was stirred at 80.degree. C. for
6 hours and then stirred at ambient temperature for 18 hours. The
colorless precipitate obtained was filtered off and the filtrate
then used directly in the procedure of Example 4 without isolation
of the product. A small amount of sample (4b) was purified by thin
layer chromatography (chloroform.methanol, 1/1) for spectroscopic
characterization.
The compounds 4a, 4c-4j were prepared at room temperature as
described above from 3a, 3c-3j, respectively.
Example 4:
General Procedure for the Preparation of
Substitute-3-N-Phenylacetylamino-2,6-Piperidinedione
The filtrate obtained in Example 3 (4b) was heated at
95.degree.-100.degree. C. for 6 hours with stirring, during which
time a colorless precipitate formed. The mixture was cooled and the
precipitate filtered off. The filtrate was stored in the
refrigerator overnight. Needle-like crystals that formed were
filtered off. The filtrate was then concentrated under vacuum at
60.degree. C. to give a syrup that was recrystallized from hot
methanol to provide 5b as a colorless crystal (5.3 g, overall yield
42.5%).
Compounds 5c-5j were prepared as described above from 4c-4j,
respectively. However, 5a was purified by a vacuum fresh column
chromatography of the syrup using chloroform/methanol (10/1) as the
eluent, to get a pale yellow crystal.
II. Activity of Cytostatic Piperidinediones
The Cytostatic activities of the active piperidinediones were
tested in three cell lines: prolactin sensitive cells (Nb2 lymphoma
cells), estrogen sensitive cells (MCF-7), and cells that are not
hormone sensitive (YAK mouse lymphoma cells). In all three cell
lines, the preferred compound, p-OH-A10, dramatically inhibited
cell growth in a dosedependent fashion. The other piperidinedione
derivatives also significantly inhibited cell growth as a function
of concentration.
Example 5
Inhibition of Prolactin Stimulated Nb2 Lymphoma Cells
The rat Nb2 lymphoma cell line, a T-cell derived lymphoma, has a
specific requirement of lactogenic hormones for growth and provides
a useful system to determine the effect of the active compounds on
hormone-dependent neoplasia. When Nb2 lymphoma cells are cultured
in media containing fetal calf serum (FCS) and horse serum (HS),
they demonstrate a doubling time of approximately 15 hours. If,
however, the cells are cultured in media devoid of FCS (lactogen
source), they enter into a quiescent state of growth. Upon addition
of exogenous prolactin (PRL), the cells resume proliferation in a
dose-dependent manner. Additionally, it has been demonstrated that
interleukin-2 (IL-2) and phorbol esters have mitogenic effects in
this cell line.
Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedione) was a
generous gift from the Burzynski Research Institute (Stafford, TX).
The A10 analogs were obtained by the synthetic scheme illustrated
in FIG. 1. Samples of A10 were analyzed for chemical composition,
purity and stability. Analyses were routinely performed on sample
solutions dissolved in dimethylformamide using a Finnigan 4023
computerized gas chromatograph-mass spectrometer under the
following conditions: injector, 300.degree. C.; source, 300.degree.
C.; oven, 265.degree. C. isotherm al; DB5 30 M capillary column;
electron energy, 70 ev; scan range, 35-450 amu. In each case, the
results showed a single chromatographic peak with the same electron
impact mass spectrum. The mass spectral fragmentation pattern was
consistent with 3-phenylacetylamino-2,6-piperidinedione (A10) (m/e
246, M+; 155, M+-C.sub.7 H.sub.7 ; 127; 118; 110; 99; 91, C.sub.7
H.sub.7 +; 84; 65; 56). While the mass spectrum of A10 was not
present in the computer library, a search revealed an expected
close match for the hydrolysis product of A10,
3-N-phenylacetylglutamine. No discernable impurities were detected
using reconstructed ion chromatograms and the ion chromatograms of
the major diagnostic ions (m/e 246, 155, and 91) possessed single
superimposable chromatographic peaks. The retention times and mass
spectra obtained in all cases were consistent with those previously
reported for A10 as well as those obtained from authentic
samples.
Penicillin/streptomycin, FCS, and Fischer's media were obtained
from Gibco Laboratories (Grand Island, N.Y.). HS was purchased from
MA Bioproducts (Walkersville, Md.). Rat prolactin (rPRL-RP3) was
supplied by NIAMDD of the National Institute of Health. Tissue
culture flasks (75 cm.sup.2), 24- and 96-well plates were obtained
from Fisher (Pittsburgh, Pa.).
Nb2 rat lymphoma cells were maintained in suspension cultures in 75
cm.sup.2 tissue culture flasks in Fischer's media supplemented with
5% horse serum (HS), 5% fetal calf serum (FCS), 10.sup.-4 M
2-mercaptoethanol, 50 units/ml penicillin, 50 .mu.g/ml
streptomycin, in an atmosphere of 5% CO.sub.2 ; 95% air at
37.degree. C.
The proliferation of NB2 lymphoma cells was determined by the
following procedure. The cells were centrifuged (300 x g, 4
minutes), washed three times in media containing only HS, and then
resuspended in the same media. Cells were then cultured for 24
hours prior to use, at which time prolactin and test compound were
added. For cell counting, 100 .mu.l aliquots of cells were added to
10 ml of isoton (Coulter Counter Electronics); the cell number was
determined in a Coulter counter (model ZM). All experiments were
performed in triplicate.
The final concentration of test compound in the culture system was
1 mg/ml. Approximately 5 mg of each of the compounds were weighed
and dissolved in heated DMSO (40.degree. C.) to give an equivalent
concentration of 200 mg/ml. An equal volume of heated 95% ethanol
was added to each compound to give a 100 mg/ml equivalent followed
by a 1:50 dilution in Fischer's media containing 5% horse serum,
pen/strp, and 2-mercaptoethanol (A10 and analog concentration - 2
mg/ml). The least soluble compound was the parent A10. It was noted
that p-OH-A10 had a yellow appearance. A volume of 400 .mu.l of
cells containing solvent were added. Following a 48 hour culture,
cell counts were made.
A comparison of the inhibitory effects of the piperidinediones on
PRL stimulation of proliferation in the NB2 lymphoma cell line (0.4
ng/ml) is provided in FIG. 2. As illustrated, p-OH-A10 provided the
greatest inhibition of lymphoma cell growth. Compounds HQH-1-45-28
(3-[N-2-chloro,6-fluorophenylacetylaminopiperidi]-2,6-dione),
HQH-1-51-29 (3-[N-4-fluorophenylacetylaminopiperidine]-2,6-dione),
and HQH-2-48-30,
(3-[N-3-fluorophenylacetylaminopiperidine]-2,6-dione) also
significantly inhibited proliferation of these cells. The
2-fluorophenyl-A10 derivative and the 2,2-difluorophenyl-A10
derivative provided some inhibition of cell growth.
Example 6:
Inhibition of MCF=7 Cells
MCF-7 cells were grown in 5% fetal calf serum and 10 .mu.g
insulin/ml in MEM supplemented media. The cells were trypsinized
during log phase growth and plated in T25 flasks at a cell number
of 20,000 cells in 2 ml media. The cells were allowed to attach
during a period of 48-72 hours. The media was then changed. The
cell numbers were counted in control flasks and 1, 2.5, 5.0 and nM
of A-10 or p-OH A-10 or control medium was added to each flask.
Each experiment was done in triplicate. The media was changed at
the end of 3 days and new aliquots of A-10 or p-OH A-10 or control
media were added. After 3 more days of cell growth, the cells were
detached from the culture flasks by adding ml of a solution of
0.05% trypsin and 0.02% EDTA. The cells were removed, and 20 ml
isoton was added and counted in a Coulter Counter. The results are
provided in Table 5 and illustrated in FIG. 3
TABLE 5 ______________________________________ Inhibition of MCF-7
Cells CELL Number (Mean .+-. SEM) Concentration (Mm) Control A-10
P-OH-A-10 ______________________________________ 1.0 73759 .+-.
3309 67342 .+-. 261 48345 .+-. 4587 2.5 79699 .+-. 2951 54164 .+-.
7409 10120 .+-. 441 5.0 60513 .+-. 5015 44961 .+-. 1947 3383 .+-.
143 10.0 66675 .+-. 4308 27654 .+-. 2266 --
______________________________________
FIG. 3 is a graph indicating the inhibitory effect of
3-[N-4-hydroxyphenylacetylaminopiperidine]-2,6-dione (p-OH A10) and
3-[N-phenylacetylaminopiperidine]-2,6-dione (A10) on MCF-7 cells
growing in log phase. As illustrated, at concentrations millimolar,
p-OH-A10 is highly active in this cell line. For example, in the
absence of test compound, MCF-7 cells growing in log phase reached
a count of approximately 80,000. When grown in log phase under the
same conditions but in the presence of 5 millimolar of A10, the
cell count was reduced to slightly less than 40,000. When grown
under the same conditions in the presence of 5 millimolar of
p-OH-A10, the cell count was reduced to less than 10,000.
Example 7
Inhibition of Mouse Lymphoma (YAK) Cells
Cells were grown in Dalbecco's modified Eagle's media in log phase.
These cell grow in suspension. Cells (22,263) were plated and
treated with 4 mM quantities of various compounds. Cell numbers
were counted after 72 hours of treatment by removing 100 .mu.l
aliquots and diluting the aliquot with 20 ml isoton. The DMSO:ETOH
concentration in all samples was 0.53%. The results are provided in
Table 6 and FIG. 4.
TABLE 6 ______________________________________ Inhibition of Mouse
Lymphoma (YAK) Cells Compound Cell Number (means .+-. SEM)
______________________________________ Control 744800 .+-. 13952
A-10 569467 .+-. 5126 PAG 543733 .+-. 7007 PA 574533 .+-. 11795
36-36 (p-OH-A-10) 34533 .+-. 3034 51-29 517600 .+-. 6526 44-11
593867 .+-. 18360 48-30 506667 .+-. 35862 45-28 466400 .+-. 18616
______________________________________
FIG. 4 is a bar chart graph indicating the inhibition of mouse
lymphoma (YAK) cell proliferation by cytostatic piperidinediones.
As illustrated, p-OH-A10 provides dramatic inhibition of the growth
of these hormone insensitive cells. Other
3-[N-phenylacetylaminopiperidine]-2,6-dione derivatives, including
the 4-fluorophenyl, the 2-fluorophenyl, the 3-fluorophenyl, and the
2-chloro,6-fluorophenyl derivatives also provided some inhibition
of cell growth.
IV. Preparation of Pharmaceutical Compositions and Mode of
Administration
As stated above, the cytostatic piperidinediones of the present
invention are useful in the study of proliferative diseases in
animal models and in vitro cell cultures. The active compounds may
also have a use in the treatment of neoplastic diseases in vivo.
Pharmaceutical compositions including these active compounds can be
prepared as described below.
The active compound or its pharmaceutically acceptable salt is
included in the pharmaceutically acceptable carrier or diluent in
an amount sufficient to exert an inhibitory effect on the growth of
the target neoplastic or proliferative cell line in vitro or in
vivo. The active materials can be administered by any appropriate
route, for example, orally, parenterally, intravenously,
intradermally, subcutaneously, intraperitoneally, or topically, in
liquid or solid form.
The active compound is included in the pharmaceutically acceptable
carrier in an amount sufficient to exert a therapeutically useful
inhibitory effect on neoplastic or proliferative cells without
serious toxic effect to healthy cells. By "inhibitory amount" is
meant an amount of active ingredient sufficient to exert a
cytostatic effect as measured by, for example, an assay such as
that described in Examples 5 through 7, or measured by blood
analysis or radiation analysis of the state of tumorigenesis in
vivo.
The concentration of active compound in the drug composition will
depend on absorption, inactivation, and excretion rates of the
active compound as well as other factors known to those of skill in
the art. It is to be noted that dosage values will also vary with
the severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions.
The active ingredient may be administered at once, or may be
divided into a number of smaller doses to be administered at
varying intervals of time.
If oral administration is desired, although not required, the
compound may be provided in a composition that protects it from the
acidic environment of the stomach. The compound can be orally
administered in combination with an antacid formulation. The
composition can also be administered in an enteric coating that
maintains its integrity in the stomach and releases the active
compound in the intestine.
Oral compositions will generally include an inert diluent or an
edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain any
of the following ingredients, or compounds of a similar nature: a
binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient such as starch or lactose, a disintegrating
agent such as alginic acid, Primogel, or corn starch; a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
When the dosage unit form is a capsule, it can contain, in addition
to material of the above type, a liquid carrier such as a fatty
oil. In addition, dosage unit forms can contain various other
materials that modify the physical form of the dosage unit, for
example, coatings of sugar, shellac, or other enteric agents.
The active compound or its pharmaceutically acceptable salt can be
administered as a component of an elixir, suspension, syrup, wafer,
chewing gum or the like. A syrup may contain, in addition to the
active compounds, sucrose as a sweetening agent and certain
preservatives, dyes, colorings and flavors.
The active compounds can also be mixed with other active materials
that do not impair the desired action, or with materials that
supplement the desired action, including other cytostatic or
anticancer compounds.
Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, lycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
If administered intravenously, preferred carriers are physiological
saline or phosphate buffered saline (PBS).
In a preferred embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations are
known to those skilled in the art. The materials can also be
obtained commercially from Nova Pharmaceutical Corporation.
Liposomal suspensions are also preferred as pharmaceutically
acceptable carriers. These may be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811 (which is incorporated herein by reference
in its entirety). For example, liposome formulations may be
prepared by dissolving appropriate lipid(s) (such as stearoyl
phosphatidyl ethanolamine, stearoyl phosphatidyl choline,
arachadoyl phosphatidyl choline, and cholesterol) in an inorganic
solvent that is then evaporated, leaving behind a thin film of
dried lipid on the surface of the container. An aqueous solution of
the active compound is then introduced into the container. The
container is then swirled by hand to free lipid material from the
sides of the container and to disperse lipid aggregates, thereby
forming the liposomal suspension.
Modifications and variations of the present invention, synthetic
piperidinediones with cytostatic activity, will be obvious to those
skilled in the art from the foregoing description. Such
modifications and variations are intended to come within the scope
of the appended claims.
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